Name: hierarchical and programmable one-pot oligosaccharide synthesis
Uploaded: 2019-09-06T13:00:00
Description: Watch this Scientific Journal Video about Hierarchical and Programmable One-Pot Oligosaccharide Synthesis at JoVE.com

This work was supported by Academia Sinica including the Summit Program, Ministry of Science and Technology [MOST 104-0210-01-09-02, MOST 105-0210-01-13-01, MOST 106-0210-01-15-02], and NSF (1664283).

1. Auto-CHO software manipulation
<ol>
	<li>Java Runtime Environment installation: make sure the Java Runtime Environment (JRE) has been installed in the device. If JRE has been installed, go to the next step, &#8220;software initialization&#8221;; otherwise, download and install JRE according to the user&#8217;s operating system found at: &#60;https://www.oracle.com/technetwork/java/javase/downloads/index.html&#62;.</li>
	<li>Software initialization: go to the Auto-CHO website at &#60;https://sites.google.com/view/aut...

<ol>
	<li>Apweiler, R., Hermjakob, H., Sharon, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=On+the+frequency+of+protein+glycosylation,+as+deduced+from+analysis+of+the+SWISS-PROT+database.">On the frequency of protein glycosylation, as deduced from analysis of the SWISS-PROT database.</a> Biochimica Et Biophysica Acta. 1473 (1), 4-8 (1999).</li><li>Sears, P., Wong, C. -. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Toward+Automated+Synthesis+of+Oligosaccharides+and+Glycoproteins.">Toward Automated Synthesis of Oligosaccharides and Glycoproteins.</a> Science. 291 (5512), 2344-2350 (2001).</li><li>Kulkarni, S. S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=&#8220;One-Pot&#8221;+Protection,+Glycosylation,+and+Protection-Glycosylation+Strategies+of+Carbohydrates.">&#8220;One-Pot&#8221; Protection, Glycosylation, and Protection-Glycosylation Strategies of Carbohydrates.</a> Chemical Reviews. 118 (17), 8025-8104 (2018).</li><li>Zhang, Z., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Programmable+One-Pot+Oligosaccharide+Synthesis.">Programmable One-Pot Oligosaccharide Synthesis.</a> Journal of the American Chemical Society. 121 (4), 734-753 (1999).</li><li>Cheng, C. -. W., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hierarchical+and+programmable+one-pot+synthesis+of+oligosaccharides.">Hierarchical and programmable one-pot synthesis of oligosaccharides.</a> Nature Communications. 9 (1), 5202 (2018).</li><li>. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> ChemDraw. , (2019).</li><li>Cheeseman, J. R., Frisch, &. #. 1. 9. 8. ;. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Predicting magnetic properties with chemdraw and gaussian. , (2000).</li><li>Yap, C. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=PaDEL-descriptor:+An+open+source+software+to+calculate+molecular+descriptors+and+fingerprints.">PaDEL-descriptor: An open source software to calculate molecular descriptors and fingerprints.</a> Journal of Computational Chemistry. 32 (7), 1466-1474 (2011).</li><li>Ceroni, A., Dell, A., Haslam, S. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+GlycanBuilder:+a+fast,+intuitive+and+flexible+software+tool+for+building+and+displaying+glycan+structures.">The GlycanBuilder: a fast, intuitive and flexible software tool for building and displaying glycan structures.</a> Source Code for Biology and Medicine. 2, 3 (2007).</li><li>Damerell, D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+GlycanBuilder+and+GlycoWorkbench+glycoinformatics+tools:+updates+and+new+developments.">The GlycanBuilder and GlycoWorkbench glycoinformatics tools: updates and new developments.</a> Biological Chemistry. 393 (11), 1357-1362 (2012).</li></ol>

The Auto-CHO software was developed for assisting chemists to proceed hierarchical and programmable one-pot synthesis of oligosaccharides5. Auto-CHO was built by Java programming language. It is a GUI software and cross-platform, which currently supports Windows, macOS, and Ubuntu. The software can be downloaded free of charge for the Auto-CHO website at &#60;https://sites.google.com/view/auto-cho/home&#62;, and its source code with MIT license can be accessed from the GitHub at &#60;https://githu...

Carbohydrates are ubiquitous in nature1,2, but their presence and mode of action remain an uncharted territory, mainly due to difficult access to this class of molecules3. Unlike automated synthesis of oligopeptides and oligonucleotides, the development of automated synthesis of oligosaccharides remains a formidable task, and progress has been relatively slow.
To tackle this problem, Wong et al. developed the first automated method for the synthesis of oligosaccharides using a programmable software program called Optimer4, which guides the selection of BBLs from a library of ~50 BBLs for sequential one-pot reactions. Each BBL was designed and synthesized with well-defined reactivity tuned by various protecting groups. Using this approach, the complexities of protecting manipulation and intermediate purification can be minimized during synthesis, which have been considered the most difficult issues to overcome in the development of automated synthesis. Despite this advance, the method is still quite restricted, as the number of BBLs is too small and the Optimer program can only handle certain small oligosaccharides. For more complex oligosaccharides that require more BBLs and multiple passes of one-pot reactions and fragment condensation, an upgraded version of the software program, Auto-CHO5, has been developed.
In Auto-CHO, more than 50,000 BBLs with defined reactivity to the BBL library have been added, including 154 synthetic and 50,000 virtual ones. These BBLs were designed by machine learning based on basic properties, calculated NMR chemical shifts6, 7, and molecular descriptors8, which affect the structure and reactivity of the BBLs. With this upgraded program and new set of BBLs available, the synthesis capacity is expanded, and as demonstrated, several oligosaccharides of interest can rapidly be prepared. It is believed that this new development will facilitate the synthesis of oligosaccharides for the study of their roles in various biological processes and their impacts on the structures and functions of glycoproteins and glycolipids. It is also thought that this work will benefit the glycoscience community significantly, given that this method is available to the research community free of charge. Synthesis of the essential human embryonic stem cell marker, SSEA-45, is demonstrated in this work.

<table>
	<tbody>
		<tr>
			<td>Acetonitrile</td>
			<td>Sigma-Aldrich</td>
			<td>75-05-8</td>
			<td></td>
		</tr>
		<tr>
			<td>Anhydrous magnesium sulfate</td>
			<td>Sigma-Aldrich</td>
			<td>7487-88-9</td>
			<td></td>
		</tr>
		<tr>
			<td>Cerium ammonium molybdate</td>
			<td>TCI</td>
			<td>C1794</td>
			<td></td>
		</tr>
		<tr>
			<td>Dichloromethane</td>
			<td>Sigma-Aldrich</td>
			<td>75-09-2</td>
			<td></td>
		</tr>
		<tr>
			<td>Drierite</td>
			<td>Sigma-Aldrich</td>
			<td>7778-18-9</td>
			<td></td>
		</tr>
		<tr>
			<td>Ethyl acetate</td>
			<td>Sigma-Aldrich</td>
			<td>141-78-6</td>
			<td></td>
		</tr>
		<tr>
			<td>Methanol</td>
			<td>Sigma-Aldrich</td>
			<td>67-56-1</td>
			<td></td>
		</tr>
		<tr>
			<td>Molecular sieves 4 &#197;</td>
			<td>Sigma-Aldrich</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>n-Hexane</td>
			<td>Sigma-Aldrich</td>
			<td>110-54-3</td>
			<td></td>
		</tr>
		<tr>
			<td>N-Iodosuccinimide</td>
			<td>Sigma-Aldrich</td>
			<td>516-12-1</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium bicarbonate</td>
			<td>Sigma-Aldrich</td>
			<td>144-55-8</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium thiosulfate</td>
			<td>Sigma-Aldrich</td>
			<td>10102-17-7</td>
			<td></td>
		</tr>
		<tr>
			<td>Toluene</td>
			<td>Sigma-Aldrich</td>
			<td>108-88-3</td>
			<td></td>
		</tr>
		<tr>
			<td>Trifluoromethanesulfonic acid</td>
			<td>Sigma-Aldrich</td>
			<td>1493-13-6</td>
			<td></td>
		</tr>
	</tbody>
</table>

hierarchical and programmable one-pot oligosaccharide synthesis

This article presents a general experimental protocol for programmable one-pot oligosaccharide synthesis and demonstrates how to use Auto-CHO software for generating potential synthetic solutions. The programmable one-pot oligosaccharide synthesis approach is designed to empower fast oligosaccharide synthesis of large amounts using thioglycoside building blocks (BBLs) with the appropriate sequential order of relative reactivity values (RRVs). Auto-CHO is a cross-platform software with a graphical user interface that provides possible synthetic solutions for programmable one-pot oligosaccharide synthesis by searching a BBL library (containing about 150 validated and &#62;50,000 virtual BBLs) with accurately predicted RRVs by support vector regression. The algorithm for hierarchical one-pot synthesis has been implemented in Auto-CHO and uses fragments generated by one-pot reactions as new BBLs. In addition, Auto-CHO allows users to give feedback for virtual BBLs to keep valuable ones for further use. One-pot synthesis of stage-specific embryonic antigen 4 (SSEA-4), which is a pluripotent human embryonic stem cell marker, is demonstrated in this work.

The Auto-CHO search result based on default parameter settings indicates SSEA-4 can be synthesized by a [2 + 1 + 3] one-pot reaction. Figure 3 shows the software screenshot of the SSEA-4 search result. When a trisaccharide reducing end acceptor is selected (Figure 3, label 1), the program shows four potential solutions for the query. The first solution has one fragment (Figure 3, label 2), and its ca...

Watch this Scientific Journal Video about Hierarchical and Programmable One-Pot Oligosaccharide Synthesis at JoVE.com

Hierarchical and Programmable One-Pot Oligosaccharide Synthesis

This protocol demonstrates how to use the Auto-CHO software for hierarchical and programmable one-pot synthesis of oligosaccharides. It also describes the general procedure for RRV determination&#160;experiments and one-pot glycosylation of SSEA-4.

This article presents a general experimental protocol for programmable one-pot oligosaccharide synthesis and demonstrates how to use Auto-CHO software for ...

We demonstrate how to use our newly-developed software, Auto-CHO, for hierarchical and programmable one-pot oligosaccharide synthesis. Auto-CHO includes algorithms on expanded building blocks, with relative reactivity values predicted through machine learning. The Auto-CHO software provides a valuable guideline for building block selection, and a hierarchical blueprint for the multiple one-pot synthesis of more complex glycans through fragment composition.The implications of this technique extend toward therapy of cancers or infectious diseases by carbohydrate-based therapeuticals. Before using Auto-CHO, please make sure the Java Runtime Environment has been installed in a PC or Mac. The visual demonstration of Auto-CHO software manipulation and the related reactivity value determination experiment will help chemists follow the protocol, and proceed with the experiment quickly.To perform software initialization of the the Java Runtime Environment, go to the Auto-CHO website and download the software according to the operating system. Currently, Auto-CHO supports Windows, macOS, and Ubuntu. The latest PDF user guide is provided on the Auto-CHO website.For Windows users, unzip the Auto-CHO Windows. zip, and double click on Auto-CHO. jar in the Auto-CHO Windows folder to start the program.Input the desired glycan structure. Choose to draw a glycan structure or read an existing structure file. To input by drawing, click on edit glycan by GlycanBuilder, or the area of click here to edit the synthetic target to draw and edit the query structure by GlycanBuilder.Linkage and chirality information should not be ignored. Click on the Globo-H, SSEA-4, or OligoLacNAc buttons to display the examples. Close the GlycanBuilder dialogue to complete editing.Define the search parameters in the parameter settings tab to get reasonable search results. Click on the okay button to enable new settings. The default setting is to search the experimental library only.If it is desired to search both the experimental and virtual libraries, select the virtual building block library tab. Select use experimental and virtual libraries, and apply filtering to display virtual building blocks with certain criteria. Experimental and virtual building blocks can work together to enhance the searching ability of Auto-CHO.Currently, Auto-CHO provides more than 50, 000 virtual building blocks, with predicted RRVs in the library. Check one or multiple desired virtual building blocks that the user would like to use for searching. Click on the show selected virtual building blocks button to show only the selected virtual building blocks.Click on the show filtered virtual building blocks button to show only virtual building blocks with certain criteria defined by the user. Click on the show all virtual building blocks button to show all available virtual building blocks, and reset the filter. Select the query structure tab and click on the search building block library button to find the one-pot synthetic solutions for the query structure.Then confirm the parameter settings. Search the result viewer. The search result is shown in the result visualization tab.The reducing end acceptors of different residue numbers are displayed in the reducing end acceptor column. Next, select a reducing end acceptor. Solutions are displayed on the synthetic solution list.Fragments are shown in the fragment list to suggest how many fragments should be used in the synthesis. The system provides detailed information of each fragment, including the RRV of the fragment, computational yield, as well as which protecting group should be deprotected for use of the fragment in the one-pot reaction. The building blocks used to assemble the selected fragment and the fragment connection information are also displayed.For experimental building blocks, view and check chemical structures of the selected building blocks in the chemical structure of building block region, and see the detailed information building block browser. In a 10-milliliter round bottom flask, combine the two thioglycoside donors, absolute methanol, and drierite in DCM. Then stir at room temperature for one hour.Take a 30-microliter aliquot of this mixture and inject the mixture into high-performance liquid chromatography in three separate injections. Measure the coefficient between the absorption and concentration of the donor molecule under the baseline separation conditions. Add a solution of 0.5 molar N-Iodosuccinimide in acetonitrile into the reaction mixture, followed by addition of a 0.1 molar trifluoromethanesulfonic acid solution.Stir the mixture at room temperature for two hours. Next, dilute the reaction mixture with four milliliters of DCM. Filter and wash the reaction with saturated aqueous sodium thiosulfate, containing 10%sodium hydrogen carbonate.Now extract the aqueous layer three times with five milliliters of DCM. Combine all organic layers, and wash with five milliliters of brine. Then dry the combined layers with approximately 200 milligrams of anhydrous magnesium sulfate.Shake the mixture mildly for 30 seconds, and filter it through a funnel with a fluted filter paper in order to remove the magnesium sulfate. Then collect the filtrate in a 25-milliliter round bottom flask. Remove the solvent, using a rotary evaporator.Dissolve the residue in one milliliter of DCM. Take a 30-microliter aliquot of this mixture, and inject it into high-performance liquid chromatography in three separate injections. Measure the concentrations of the remaining donors by HPLC, under the same separation conditions.Measure the relative reactivity. Based on the relative reactivity value of DR4, the relative reactivity value of DX1 is three. The Auto-CHO search result based on default parameter settings indicates SSEA-4 can be synthesized by a two plus one plus three one-pot reaction.When a trisaccharide-reducing end acceptor is selected, the program shows four potential solutions for the query. The first solution has one fragment, and its calculated yield is about 94%The fragment can be synthesized by two building blocks. The RRV of the first disaccharide building block is 1462, and the RRV of the second monosaccharide is 32.0.The chemical structure of the first suggested building block used in the one-pot reaction is also shown. The one-pot experiment shows that SSEA-4 can be successfully synthesized in 43%yield by this suggestion. SSEA-4 can be synthesized by three units suggested by Auto-CHO.These units include silele disaccharide building block one, monosaccharide building block two, and reducing end acceptor three. For parameter settings, we suggest to set parameters with stricter criteria at the beginning. For the selection of the building block library, we suggest to search the experimental library only at first.Through this demonstration, we hope more important glycans, such as tumor-associated carbohydrate antigens, can be synthesized by the one-pot approach for further studies. Following this procedure, synthesis of all such antigens can be performed in order to design carbohydrate-based cancer vaccines. We also hope that artificial intelligence and computer algorithms can facilitate automated glycan synthesis to benefit disease treatment and prevention.

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